CN111592679A - Novel nano hydrogel for promoting growth of lactic acid bacteria and preparation method thereof - Google Patents
Novel nano hydrogel for promoting growth of lactic acid bacteria and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a novel nano hydrogel for promoting the growth of lactic acid bacteria and a preparation method thereof, which comprises the steps of firstly, uniformly mixing a polymer containing bisphosphonate, a yolk low-density lipoprotein solution and sodium carboxymethyl starch, and reacting under the action of glutaraldehyde to obtain the nano hydrogel; and then soaking the nano hydrogel in a bisphosphonate buffer salt solution and a manganese sulfate solution in sequence to obtain the target nano hydrogel. The nano particles have a certain space volume, expand the growth range of thalli and play a role in separating the growth of thalli; the added yolk low-density lipoprotein belongs to a nano-scale spherical macromolecular substance, contains lipid and protein, and provides nutrition required for the growth of lactic acid bacteria; in addition, the manganese ions added into the nano hydrogel have an obvious effect on promoting the growth of lactic acid bacteria. Compared with the traditional method for culturing the lactic acid bacteria, the material prepared by the invention not only provides a three-dimensional growth space for the bacteria, but also effectively promotes the growth of the bacteria.
Description
Technical Field
The invention belongs to the field of high polymer materials, and relates to novel nano hydrogel for promoting the growth of lactic acid bacteria and a preparation method thereof.
Background
Lactic acid bacteria are not only ideal materials for researching biochemistry, heredity, molecular biology and genetic engineering, but also have extremely high application value in important fields closely related to human life, such as industry, farming and animal husbandry, food, medicine and the like. At present, MRS solid culture medium and liquid culture medium are the most common methods for culturing lactic acid bacteria, the lactic acid bacteria growing on a plane are cultured on the solid culture medium, the bacterial colony is small, the growth is slow, and the growth environment is limited; lactic acid bacteria can grow quickly in a liquid fermentation medium, but a carrier capable of being attached is lacked in the growth process, most of the bacteria are in a free state, and finally the collection process is tedious and high in cost. In addition, the growth of the lactic acid bacteria needs the supply of trace elements such as manganese ions, but the simple manual addition is difficult to control the amount, and the addition cost is high depending on automatic equipment. Lipid and protein are also important nutrient components for promoting the growth of lactic acid bacteria, most of materials for preparing the carrier are petroleum-based polyolefin derivatives at present, and few reports are provided for preparing a main body framework and a key embedding object of the carrier by taking the lipid and the protein as raw materials. Therefore, if a carrier material capable of promoting the growth of lactic acid bacteria can be developed, it is very important to provide comprehensive nutrients and have the function of slowly supplying trace elements.
Disclosure of Invention
The invention aims to solve the problems that the growth space of the lactobacillus in the lactobacillus culture method in the prior art is insufficient, the lactobacillus grows slowly, no attachable carrier exists, the addition control of trace elements is difficult, and the like. Aiming at the series of problems, a novel nano hydrogel for promoting the growth of lactic acid bacteria is developed, and a bisphosphonate polymer capable of adsorbing manganese ions, yolk low-density lipoprotein and sodium carboxymethyl starch are selected as raw materials for preparing the hydrogel; the three raw materials react with other raw materials to obtain the novel carboxymethyl starch sodium nano hydrogel capable of promoting the growth of the lactic acid bacteria.
In order to solve the technical problem, the technical scheme adopted by the invention is as follows:
a preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria comprises the steps of firstly synthesizing a bisphosphonate-containing polymer with a manganese ion adsorption function, dissolving the bisphosphonate-containing polymer by a high-speed shearing method to obtain a yolk low-density lipoprotein solution and sodium carboxymethyl starch, finally uniformly mixing the substances obtained in the two steps, and reacting the substances under the action of glutaraldehyde to obtain the nano hydrogel; soaking the nano hydrogel in manganese ion-containing solution containing substances for promoting the growth of the lactic acid bacteria, and freeze-drying to obtain the nano hydrogel for promoting the growth of the lactic acid bacteria.
The method comprises the following specific steps:
(1) dissolving 1-10g of acrylate bisphosphonate, 1-10g of hydroxyethyl methacrylate, 1-10ml of acrylic acid and 0.1-1.6g of azobisisobutyronitrile into 60-150ml of anhydrous tetrahydrofuran, ethanol or methanol, reacting for 15-30min under the protection of nitrogen, then placing a reaction bottle in an oil bath kettle at 60-90 ℃ for reacting for 6-24h, condensing and refluxing, and performing reduced pressure distillation after the reaction is finished to remove a solvent and unreacted monomers, thus obtaining a polymer containing bisphosphonate;
(2) dissolving 1-15g of yolk low-density lipoprotein in 10-100ml of deionized water by a high-speed shearing method to prepare a yolk low-density lipoprotein solution;
(3) dissolving 1-30g of sodium carboxymethyl starch in 10-100ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution;
(4) weighing 1-10g of the polymer containing bisphosphonate prepared in the step (1) and 1-10ml of the sodium carboxymethyl starch solution prepared in the step (3), adding the mixture into 1-5ml of the egg yolk low-density lipoprotein solution prepared in the step (2), violently stirring for 20-30h, then dripping the mixture into 20-40ml of an acetone solution containing 5-10 wt% of boric acid through an injector under the stirring of 200-300rpm, aging the solution for 20-25h after finishing dripping, adding 1-6ml of glutaraldehyde and 1mol/L of hydrochloric acid into the mixture, and incubating for 7-10h at 40-60 ℃ to obtain nano hydrogel;
(5) soaking the nano hydrogel obtained in the step (4) in 3-10 wt% of 100-ml manganese sulfate, manganese dichloride or manganese nitrate solution for 1-10h by using a dipping method, washing off free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the nano hydrogel for promoting the growth of lactic acid bacteria.
The preparation method of the acrylate bisphosphonate in the step (1) comprises the following steps: dissolving 0.1-1mol of N-acryloyloxy succinimide and 0.01-0.1mol of pamidronic acid disodium salt in 100ml of NaOH solution with the pH value of 8.0 together to perform stirring reaction; reacting for 12-48h at room temperature, adding absolute ethyl alcohol, centrifuging, collecting precipitate, and washing with absolute ethyl alcohol for several times to obtain acrylate bisphosphonate.
A novel nano hydrogel for promoting the growth of lactobacillus prepared by the preparation method.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides favorable growth space and microenvironment for the growth of thalli by taking a proper nano hydrogel body as a carrier. In addition, the lactobacillus collecting device is small in size, non-toxic, free of special treatment during lactobacillus collection and good in operability.
2. The yolk low-density lipoprotein used by the invention is taken as a raw material, so that the carrier directly has the function of providing protein and lipid required by the growth of lactic acid bacteria, and the petroleum-based polyolefin derivative which is taken as the raw material of the carrier at present is replaced, thereby facilitating the source of the raw material and saving the non-renewable petroleum resource.
3. The invention can realize the controllable slow release of manganese ions, which can provide trace elements for the growth of lactobacillus and promote the growth of lactobacillus.
Detailed Description
The present invention will be further illustrated by the following examples.
Example 1
A preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria comprises the following steps:
1) 0.1mol of N-acryloyloxysuccinimide and 0.01mol of pamidronic acid disodium salt were dissolved together in 100ml of NaOH solution (pH 8.0) and stirred for reaction. After reacting for 24 hours at room temperature, adding absolute ethyl alcohol, centrifuging and collecting precipitate, and washing with the absolute ethyl alcohol for a plurality of times to prepare the acrylate bisphosphonate.
2) 1g of acrylate bisphosphonate, 1g of hydroxyethyl methacrylate, 1ml of acrylic acid and 0.16 g of azobisisobutyronitrile are dissolved in 60ml of anhydrous tetrahydrofuran, the mixture reacts for 20min under the protection of nitrogen, then a reaction bottle is placed in an oil bath kettle at 60 ℃ for reaction for 12 hours, the mixture is condensed and refluxed, and after the reaction is finished, reduced pressure distillation is carried out to remove the solvent and unreacted monomers, thus obtaining the polymer containing bisphosphonate.
3) The yolk low density lipoprotein solution was prepared by dissolving 1g of yolk low density lipoprotein in 10ml of deionized water by high speed shearing.
4) 1g of sodium carboxymethyl starch is dissolved in 10ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution.
5) Weighing 1g of polymer containing bisphosphonate 1g and 1ml of sodium carboxymethyl starch solution, adding the solution into 1ml of yolk low-density lipoprotein solution, violently stirring for 24h, then dripping the mixture into 20ml of acetone solution containing 5 wt% of boric acid through an injector under the stirring of 250rpm, aging the solution for 24h after the dripping is finished, then adding 1ml of glutaraldehyde and 1mol/L of hydrochloric acid 1ml into the mixture, and incubating for 8h at 50 ℃ to obtain the sodium hydroxymethyl nano hydrogel.
6) Soaking the finally obtained nano hydrogel in 5 wt% of 100ml manganese nitrate solution for 5h by a dipping method, washing away free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the final nano hydrogel for promoting the growth of the lactic acid bacteria.
Example 2
A preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria comprises the following steps:
1) 0.5mol of N-acryloyloxysuccinimide and 0.05mol of pamidronic acid disodium salt were dissolved together in 500ml of NaOH solution (pH 8.0) and stirred for reaction. After reacting for 36 hours at room temperature, adding absolute ethyl alcohol, centrifuging and collecting precipitate, and washing with absolute ethyl alcohol for several times to obtain the acrylate bisphosphonate.
2) 5g of acrylate bisphosphonate, 5g of hydroxyethyl methacrylate, 5ml of acrylic acid and 0.1 g of azobisisobutyronitrile are dissolved in 100ml of methanol and reacted for 15min under the protection of nitrogen, then the reaction bottle is placed in an oil bath kettle at 80 ℃ for reaction for 18 h and condensed and refluxed, and after the reaction is finished, reduced pressure distillation is carried out to remove the solvent and unreacted monomers, thus obtaining the polymer containing the bisphosphonate.
3) The yolk low density lipoprotein solution was prepared by dissolving 8g of yolk low density lipoprotein in 50ml of deionized water by high speed shearing.
4) 15g of sodium carboxymethyl starch is dissolved in 50ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution.
5) Weighing 5g of polymer containing bisphosphonate and 5ml of sodium carboxymethyl starch solution, adding the polymer containing bisphosphonate and 5ml of sodium carboxymethyl starch solution into 2ml of egg yolk low-density lipoprotein solution, stirring for 20h vigorously, then dripping the mixture into 30ml of acetone solution containing 7 wt% of boric acid through a syringe under the stirring of 200rpm, aging the solution for 20h after the dripping is finished, adding 3ml of glutaraldehyde and 2ml of 1mol/L hydrochloric acid into the mixture, and incubating for 7h at 40 ℃ to obtain the sodium hydroxymethyl nano hydrogel.
6) Soaking the finally obtained nano hydrogel in 250ml of manganese dichloride solution with the weight percent of 3 for 1h by a dipping method, washing away free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the nano hydrogel for finally promoting the growth of the lactic acid bacteria.
Example 3
A preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria comprises the following steps:
1) 0.8mol of N-acryloyloxysuccinimide and 0.08mol of pamidronic acid disodium salt were dissolved together in 800ml of NaOH solution (pH 8.0) and stirred for reaction. After reacting for 12 hours at room temperature, adding absolute ethyl alcohol, centrifuging and collecting precipitate, and washing with the absolute ethyl alcohol for a plurality of times to prepare the acrylate bisphosphonate.
2) Dissolving 8g of acrylate bisphosphonate, 8g of hydroxyethyl methacrylate, 8ml of acrylic acid and 1g of azobisisobutyronitrile into 120ml of anhydrous tetrahydrofuran, reacting for 30min under the protection of nitrogen, then placing a reaction bottle in an oil bath kettle at 90 ℃ for reacting for 24 hours, condensing and refluxing, and after the reaction is finished, carrying out reduced pressure distillation to remove the solvent and unreacted monomers, thus obtaining the polymer containing bisphosphonate.
3) The yolk low density lipoprotein solution was prepared by dissolving 15g of yolk low density lipoprotein in 80ml of deionized water by high speed shearing.
4) 25g of sodium carboxymethyl starch is dissolved in 80ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution.
5) Weighing 8g of polymer containing bisphosphonate and 8ml of sodium carboxymethyl starch solution, adding the polymer containing bisphosphonate and 8ml of sodium carboxymethyl starch solution into 4ml of egg yolk low density lipoprotein solution, stirring for 30h vigorously, then dropping the mixture into 35ml of acetone solution containing 8 wt% of boric acid through a syringe under the stirring of 300rpm, aging the solution for 25h after the dropping is finished, adding 6ml of glutaraldehyde and 4ml of 5mol/L hydrochloric acid into the mixture, and incubating for 10h at 60 ℃ to obtain the sodium hydroxymethyl nano hydrogel.
6) Soaking the finally obtained nano hydrogel in 400ml of 8 wt% manganese sulfate solution for 8h by using a dipping method, washing away free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the nano hydrogel for finally promoting the growth of lactic acid bacteria.
Example 4
A preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria comprises the following steps:
1) 1mol of N-acryloyloxysuccinimide and 0.1mol of pamidronic acid disodium salt were dissolved together in 1000ml of NaOH solution (pH 8.0) and stirred for reaction. After reacting for 48 hours at room temperature, adding absolute ethyl alcohol, centrifuging and collecting precipitate, and washing with the absolute ethyl alcohol for a plurality of times to prepare the acrylate bisphosphonate.
2) Dissolving 10g of acrylate bisphosphonate, 10g of hydroxyethyl methacrylate, 10ml of acrylic acid and 1.6g of azobisisobutyronitrile into 150ml of ethanol, reacting for 20min under the protection of nitrogen, then placing a reaction bottle in an oil bath kettle at 90 ℃ for reacting for 24 hours, condensing and refluxing, and after the reaction is finished, carrying out reduced pressure distillation to remove the solvent and unreacted monomers, thus obtaining the polymer containing bisphosphonate.
3) The yolk low density lipoprotein solution was prepared by dissolving 15g of yolk low density lipoprotein in 100ml of deionized water by high speed shearing.
4) 30g of sodium carboxymethyl starch is dissolved in 100ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution.
5) Weighing 10g of polymer containing bisphosphonate 10g and 10ml of sodium carboxymethyl starch solution, adding the polymer into 5ml of egg yolk low density lipoprotein solution 5ml, violently stirring for 30h, then dropping the mixture into 40ml of acetone solution containing 10 wt% of boric acid through an injector under the stirring of 250rpm, aging the solution for 24h after the dropping is finished, then adding 6ml of glutaraldehyde and 5ml of 1mol/L hydrochloric acid into the mixture, and incubating for 10h at 60 ℃ to obtain the sodium hydroxymethyl nano hydrogel.
6) Soaking the finally obtained nano hydrogel in 10 wt% of 500ml manganese sulfate solution for 10h by using a dipping method, washing away free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the nano hydrogel for finally promoting the growth of lactic acid bacteria.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (4)
1. A preparation method of novel nano hydrogel for promoting growth of lactic acid bacteria is characterized by comprising the following steps: firstly, synthesizing a polymer containing bisphosphonate with a manganese ion adsorption function, dissolving the polymer by a high-speed shearing method to obtain an egg yolk low-density lipoprotein solution and sodium carboxymethyl starch, and finally, uniformly mixing the substances obtained in the two steps, and reacting the substances under the action of glutaraldehyde to obtain nano hydrogel; soaking the nano hydrogel in manganese ion-containing solution containing substances for promoting the growth of the lactic acid bacteria, and freeze-drying to obtain the nano hydrogel for promoting the growth of the lactic acid bacteria.
2. The method of claim 1, comprising the steps of:
(1) dissolving 1-10g of acrylate bisphosphonate, 1-10g of hydroxyethyl methacrylate, 1-10ml of acrylic acid and 0.1-1.6g of azobisisobutyronitrile into 60-150ml of anhydrous tetrahydrofuran, ethanol or methanol, reacting for 15-30min under the protection of nitrogen, then placing a reaction bottle in an oil bath kettle at 60-90 ℃ for reacting for 6-24h, condensing and refluxing, and performing reduced pressure distillation after the reaction is finished to remove a solvent and unreacted monomers, thus obtaining a polymer containing bisphosphonate;
(2) dissolving 1-15g of yolk low-density lipoprotein in 10-100ml of deionized water by a high-speed shearing method to prepare a yolk low-density lipoprotein solution;
(3) dissolving 1-30g of sodium carboxymethyl starch in 10-100ml of deionized water by a high-speed shearing method to obtain a sodium carboxymethyl starch solution;
(4) weighing 1-10g of the polymer containing bisphosphonate prepared in the step (1) and 1-10ml of the sodium carboxymethyl starch solution prepared in the step (3), adding the mixture into 1-5ml of the egg yolk low-density lipoprotein solution prepared in the step (2), violently stirring for 20-30h, then dripping the mixture into 20-40ml of an acetone solution containing 5-10 wt% of boric acid through an injector under the stirring of 200-300rpm, aging the solution for 20-25h after finishing dripping, adding 1-6ml of glutaraldehyde and 1mol/L of hydrochloric acid into the mixture, and incubating for 7-10h at 40-60 ℃ to obtain nano hydrogel;
(5) soaking the nano hydrogel obtained in the step (4) in 3-10 wt% of 100-ml manganese sulfate, manganese dichloride or manganese nitrate solution for 1-10h by using a dipping method, washing off free ions and unadsorbed ions on the surface of the hydrogel by using ethanol, and freeze-drying to obtain the nano hydrogel for promoting the growth of lactic acid bacteria.
3. The method of claim 2, wherein: the preparation method of the acrylate bisphosphonate in the step (1) comprises the following steps: dissolving 0.1-1mol of N-acryloyloxy succinimide and 0.01-0.1mol of pamidronic acid disodium salt in 100ml of NaOH solution with the pH value of 8.0 together to perform stirring reaction; reacting for 12-48h at room temperature, adding absolute ethyl alcohol, centrifuging, collecting precipitate, and washing with absolute ethyl alcohol for several times to obtain acrylate bisphosphonate.
4. A novel nano-hydrogel for promoting the growth of lactic acid bacteria prepared by the preparation method according to any one of claims 1 to 3.
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